Abstract

BACKGROUND:

Human eosinophil-derived neurotoxin (edn) and eosinophil cationic protein (ecp) are members of a subfamily of primate ribonuclease (rnase) genes. Although they are generated by gene duplication event, distinct edn and ecp expression profile in various tissues have been reported.

RESULTS:

In this study, we obtained the upstream promoter sequences of several representative primate eosinophil rnases. Bioinformatic analysis revealed the presence of a shared 34-nucleotide (nt) sequence stretch located at -81 to -48 in all edn promoters and macaque ecp promoter. Such a unique sequence motif constituted a region essential for transactivation of human edn in hepatocellular carcinoma cells. Gel electrophoretic mobility shift assay, transient transfection and scanning mutagenesis experiments allowed us to identify binding sites for two transcription factors, Myc-associated zinc finger protein (MAZ) and SV-40 protein-1 (Sp1), within the 34-nt segment. Subsequent in vitro and in vivo binding assays demonstrated a direct molecular interaction between this 34-nt region and MAZ and Sp1. Interestingly, overexpression of MAZ and Sp1 respectively repressed and enhanced edn promoter activity. The regulatory transactivation motif was mapped to the evolutionarily conserved -74/-65 region of the edn promoter, which was guanidine-rich and critical for recognition by both transcription factors.

CONCLUSION:

Our results provide the first direct evidence that MAZ and Sp1 play important roles on the transcriptional activation of the human edn promoter through specific binding to a 34-nt segment present in representative primate eosinophil rnase promoters.

The role of the conserved regions -74/-65 and -62/-48 in the 34-nt region in transcriptional activity in HepG2 cells. (A) Expression of edn and ecp mRNA in HepG2 and HL-60 clone 15 cells. Total RNA of HepG2 and HL-60 clone 15 were isolated and reverse-transcripted into cDNA. Specific primers for ecp or edn were used to amplify the cDNA. The PCR products were electrophoresed on a 2.0% agarose gel and stained with ethidium bromide. (B) HepG2 cells were transfected with the luciferase reporter plasmid pGL3 basic or the same vector containing edn or ecp upstream sequences with or without the 34-nt segment. (C) HepG2 cells were transfected with the luciferase reporter plasmid pGL3 basic or pGL3-edn, pGL3-ednΔ (-81/-48), pGL3-edn mut (-74/-65), pGL3-edn mut (-62/-48), respectively. The promoter activities were measured using the luciferase assay system. The average values of promoter activities were calculated as described in Methods and obtained from five independent experiments. The difference between the two groups is statistically significant (P < 0.05), as determined by the Wilcoxon Rank Sum test.

Identification of the transcription factors that recognize the wild type 34-nt segment. (A) In the presence of anti-MAZ, EMSA gel shift reactions were preincubated with of monoclonal anti-MAZ (lane 3) or control antibody (anti-mouse IgG; lane 2) for 30 min prior to the addition of the labeled wild type 34-nt probe. The position of the major protein/DNA complex and free probe is indicated by an arrowhead and F, respectively. (B) Purified recombinant GST-MAZ (lanes 1 to 4) and GST-MAZΔF34 (lanes 5 to 8) were expressed, purified, and used for EMSA with the wild type 34-nt probe.

Identification of the putative regulatory elements for MAZ binding within the wild type 34-nt segment. (A) Identification of the MAZ-responsive core sequence. Scanning mutagenesis EMSA was used to define the binding site of the putative regulatory region. 32P-labeled wild type 34-nt segment was used for all experiments in the presence of 200-fold molar excess of unlabeled oligonucleotide competitor as indicated. The sequences of the mutant oligonucleotides m1 to m21 are shown in supplementary Table . (B) Summary of the DNA-binding activity of nuclear extract proteins to various mutant competitors to the wild type 34-nt segment. The stars above the sequence shown at the bottom indicate the nucleotides crucial for MAZ binding, and the dark circles below the sequence indicate the nucleotides that are less important for MAZ binding. (C) Western blot analysis of MAZ in nuclear extracts (50 μg protein) isolated from pcDNA3- and pcDNA3/MAZ-transfected HepG2 cells (lanes 1 and 2, respectively). (D) Nuclear extracts were prepared from HepG2 cells and incubated with biotinylated oligonucleotide from the 34-nt region of the edn promoter. The samples were separated by 8% SDS-PAGE and immunoblotted for MAZ. Relative densitometric quantitation of bands in lanes 1–5 are as follows: biotin control, wild type, mutant -62/-48, mutant -74/-65, mutant -62/-48 & -74/-65. The difference between the two groups is statistically significant (P < 0.05), as determined by the Wilcoxon Rank Sum test.

Sp1 and MAZ competition for the same binding site within the 34-nt oligonucleotide in vitro. (A) Autoradiograms from EMSA using a constant amount of Sp1 protein and increasing quantities of GST-MAZ protein. (B) Autoradiograms from EMSA using a constant amount of GST-MAZ protein and increasing quantities of Sp1 protein. The lower panels show quantitation of the bands in each fraction.

Binding of Sp1 and MAZ to the 34-nt region in the edn promoter in vivo. (A) Cells were fixed by 1% formaldehyde, and the genomic DNA was sonicated until the size was approximately 1000 bp. The samples were then analyzed by the ChIP assay for Sp1, using IgG as a negative control. The samples were subjected to 30 cycles of PCR and separated on a 2% agarose gel. (B) ChAP assays were performed using HepG2 cells transfected with pcDNA3/MAZ-6H, pcDNA3/MAZΔF34-6H, or empty pcDNA plasmid. Two days after transfection, cells were scraped, cross-linked and sonicated, and the DNA-protein complexes were incubated with nickel resin slurry for 1 hr at 4°C. The affinity precipitated DNA/protein complexes were eluted by 1 M imidazole and subjected PCR amplification. Input DNA served as a positive control.

Differential regulatory roles of MAZ and Sp1 on transactivation through binding to the 34-nt sequence motif of the edn promoter. (A) Cotransfections and luciferase assays were performed with HepG2 cells in the presence of pcDNA3, pcDNA3/MAZ or pcDNA3/MAZΔF34. (B) Similar to (A), but cotransfection was performed with pcDNA3 or pcDNA3/Sp1. (C) Cells were transfected with 40 pmol/well Sp1/RNAi1, Sp1/RNAi2, or RNAi control. Forty-eight hours later, cells were harvested and protein lysates were separated by 8% SDS-PAGE and transferred to a PVDF membrane that was probed for Sp1 or actin. (D) Cotransfections and luciferase assays using wt and Δ (-81/-48) edn promoter constructs were carried out with HepG2 cells in the presence of control RNAi, Sp1/RNAi1 or Sp1/RNAi2. The difference between the two groups is statistically.